It is indispensable to many crops to keep their qualities for a long time after harvest by storing them at a cold place or the like. However, it is known that if potato tubers are stored at a low temperature, accumulation of reducing sugar occurs, which is called low temperature sweetening, and the generated reducing sugar causes a coloring reaction (Maillard reaction) during processing them to French fried potatoes, potato chips or the like, so that the values of the commercial products are largely decreased. It is also known that fruits are softened by generation of ethylene. To overcome these problems, post-harvest physiology is now widely studied throughout the world.
In specifically expressing a foreign gene in potato tubers, the promoter of the gene for a storage protein patatin has been widely used in the world (EMBO J. 8(1): 23-29, 1989, Plant Mol. Biol. 12:41-50, 1989, Bio/Technology 12: 1101-1105, 1994 and so on). Expression of patatin gene increases with the development of the tuber. However, in the stage in which the tubers are developed, various metabolic systems such as that for converting reducing sugar to starch are activated. Therefore, it is possible that the gene ligated to the patatin promoter may disturb a metabolic system, which may lead to decrease in the yield. To avoid such a problem, it is thought necessary to isolate and utilize a promoter which efficiently promotes the expression of genes only in tubers stored at low temperatures and hardly promotes the expression of genes in the plant under normal conditions.
A number of types of cold-inducible genes from prokaryotic and eukaryotic organisms have been reported (There are reviews including Tissue Culture, 19(10): 357-361, 1993). Cold-inducible genes have also been isolated from potato tubers (Plant Physiol. 104: 445-452, 1994). It has been reported that an osmotin-like gene is isolated from a wild potato species, and that its expression is induced at low temperatures (Plant Mol. Biol. 21:729-735, 1993). The above-mentioned report is the only one which reports isolation of cold-inducible genes from potato tubers.
Five types of cold-inducible genes (cDNAs) have been isolated from potato tubers (Plant Physiol. 104: 445-452, 1994). Two of them have similarities to genes for known small heat-shock proteins, or to genes for cold-inducible proteins and ABA-inducible proteins of other plants. Other three genes have not been sequenced. The genomic DNAs (including promoters) of these cDNAs have not been isolated. It is thought that all of these genes quickly (within one week) respond to low temperatures.
Needless to say, promoters of the above-described known genes can be applied to crops (such as potato) which are required to be stored at a low temperature. However, it is not known whether these promoters (Plant Physiol. 104: 445-452, 1994 or the like) induce high expression in the desired organ alone (it is possible that these promoters are induced also in other organs at low temperatures). Further, genes controlled by these promoters may be expressed at normal temperature to a certain degree. Thus, it is not known whether efficient expression may be carried out only in the organs to be stored at a low temperature, such as potato tubers. Further, although potato tubers are stored for as long as several months, it is not known whether the above-described promoters function for such a long time (Plant Physiol. 104: 445-452, 1994. In this reference, the promoter activities are tested for only about one month).